NK cells are a subpopulation of lymphocytes whose unique receptors facilitate the detection of infected, transformed, or "stressed" cells. This immune recognition subsequently leads to the development of NK cell- mediated cytotoxicity or the generation of cytokines and chemokines that activate other components of the immune system. Importantly, the signaling motifs used by many activating receptors on human NK cells (e.g. NKG2D and 2B4) are substantially different from those used by traditional immune recognition receptors on other hematopoietic cell types. In addition, although cell-mediated killing is a central function for NK cells, the actual downstream regulatory events controlling the terminal phases of MTOC and granule polarization, granule tethering to the plasma membrane, and the fusion of the granule-plasma membrane lipid bilayers are poorly understood. Based on the preliminary data included in this proposal, we hypothesize that: (a) NKG2D- initiated activation is critically influenced by the binding of the adaptor molecule GADS;(b) SAP-binding to the 2B4 receptor facilitates a required direct interaction with ITAM-containing receptor complexes on NK cells;(c) The actin-nucleating formins FMNL1 and DIA1 regulate the initial phase of MTOC polarization that precedes the final translocation of the vesicles to the plams membrane;(d) The dynamin family member Dyn2 regulates the terminal phases of granule exocytosis in a GTPase-independent manner;(e) A physical interaction between Dyn2 and syntaxin 4 regulates the docking of granules to the plasma membrane;and (f) The SNARE protein syntaxin 11 is required for granule release from cytotoxic lymphocytes. In order to test these hypotheses we will: (1) Evaluate the differential signaling pathways utilized by the non-ITAM containing NK receptors NKG2D, and 2B4;and (2) Identify and characterize the roles of formins, dynamin 2 and SNARE proteins in the regulation of granule secretion during cell-mediated cytotoxicity. Together these studies will provide an experimental basis for understanding the molecular events that are involved in the regulation of NK cell effector functions, and will, in a broader context, advance our understanding of fundamental processes in cellular activation, transmembrane signaling and granule exocytosis. PUBLIC HEALTH RELEVANCE Completion of the outlined experiments will provide new insights into the mechanisms regulating the ability of cytotoxic lymphocytes to mediate effective immunity. More broadly, these studies will provide a basis for therapeutically targeting specific cellular processes in order to enhance antitumor and antiviral immunity.